This invention relates to a method of modifying the genome of an entero-invasive wild strain of Shigella so that the strain cannot substantially invade cells and tissues of an infected host and cannot spread substantially within infected cells and between infected and non-infected cells of the host and cannot produce toxins which will kill substantial numbers of the hosts' cells. This invention particularly relates to such a modified strain of Shigella which can be used to immunize a host against the wild strain of Shigella. 
Shigellosis or bacillary dysentery is a disease that is endemic throughout the world. The disease presents a particularly serious public health problem in tropical regions and developing countries where Shigella dysenteriae 1 and S. flexneri predominate. In industrialized countries, the principal etiologic agent is S. sonnei although sporadic cases of shigellosis are encountered due to S. flexneri, S. boydii and certain entero-invasive Escherichia coli. 
The primary step in the pathogenesis of bacillary dysentery is invasion of the human colonic mucosa by Shigella (23). Mucosal invasion encompasses several steps which include penetration of the bacteria into epithelial cells, intracellular multiplication, killing of host cells, and final spreading to adjacent cells and to connective tissue (9, 41, 55, 56). The overall process which is usually limited to the mucosal surface leads to a strong inflammatory reaction which is responsible for abscesses and ulcerations (23, 41, 55).
Even though dysentery is characteristic of shigellosis, it may be preceded by watery diarrhea. Diarrhea appears to be the result of disturbances in colonic reabsorption and increased jejunal secretion whereas dysentery is a purely colonic process (20, 41). Systemic manifestations may also be observed in the course of shigellosis, mainly in the cases due to S. dysenteriae 1. These include toxic megacolon, leukemoid reactions and hemolytic-uremic syndrome (“HUS”). The latter is a major cause of mortality from shigellosis in developing areas (11, 22, 38).
The role of Shiga-toxin produced at high level by S. dysenteriae 1 (6) and Shiga-like toxins (“SLT”) produced at low level by S. flexneri and S. sonnei (19, 30) in the four major stages of shigellosis (i.e., invasion of individual epithelial cells, tissue invasion, diarrhea and systemic symptoms) is not well understood. For review see O'Brien and Holmes (32). Plasmids of 180–220 kilobases (“kb”) are essential in all Shigella species for invasion of individual epithelial cells (41, 42, 44). This includes entry, intracellular multiplication and early killing of host cells (4, 5, 46). The role of Shiga-toxin and SLT at this stage is unclear. They do not appear to play a crucial role in intracellular multiplication and early killing (4, 12, 46). However none of the experiments which have been carried out has compared isogenic mutants in a relevant cell assay system. Recent evidence indicates that Shiga-toxin is cytotoxic for primary cultures of human colonic cells (27). Tissue invasion requires additional chromosomally encoded products among which are smooth lipopolysaccharides (“LPS”) (44, 57), the non-characterized product of the Kcp locus (8, 44), and aerobactin (24, 28). A region of the S. flexneri chromosome necessary for fluid production in rabbit ileal loops has been localized to the rha-mtl regions and near the lysine decarboxylase locus (44). However, no evidence has been adduced to show that the ability to cause fluid accumulation is due to the SLT of S. flexneri. Thus, the role of Shiga-toxin in causing the systemic complications of shigellosis is still hypothetical. However, Shiga-toxin can mediate vascular damage since capillary lesions observed in HUS resemble those observed in cerebral vessels of animals injected with this toxin (1, 2, 22).
A mutant which lacks Shiga-toxin or SLT could indicate the role of these toxins in the disease process. S. dysenteriae 1, which produces the highest amount of this cytotoxin, could be transformed into such a Shiga-toxin negative mutant (“Tox−”) and could serve best to indicate the role of the toxin—despite Sekizaki et al's (48) having obtained such a mutant which appeared as invasive in the HeLa cell assay and the Sereny test (49) as the wild strain. More importantly, such a Tox− mutant could be used to make a mutant which could not invade, and then multiply substantially within, cells of a host and also could not spread substantially within the host's infected cells and from there to the host's uninfected cells and also could not produce toxins which would kill subtantial numbers of infected, as well as uninfected, host cells. As a result, the Tox− mutant could be used to immunize a host against a wild strain of the Shigella. 